This application will develop robust techniques to manipulate gene expression and investigate gene function in embryos of an important new metazoan model system, Nematostella vectensis. Surprising new results from total genome sequencing and EST analyses of N. vectensis indicate that vertebrates share greater similarities in genome content with cnidarians like N. vectensis than either do with flies or nematodes. This revelation casts doubt on the significance that derived genetic model systems may have on human biology and thus provides the opportunity to understand the ancestral relationships between genetic variation and morphological complexity. The crucial phylogenetic position of N. vectensis will allow an understanding of the differences in the molecular control of developmental patterning seen in vertebrates, but absent in derived ecdysozoan genetic models systems, providing important insight into the ancient molecular patterning mechanism deployed before the radiation of vertebrate lineages. Being able to experimentally dissect molecular pathways in these animals is crucial to understanding the origin of variation in genetic regulatory networks involved in normal human health and disease. This work takes advantage of the complete genome sequencing of N. vectensis by the Joint Genome Institute (Department of Energy) and will provide additional technical and methodological background for future experiments in the field. This application will substantially enhance our ability to launch a long term research program for gene discovery and functional investigations of genetic regulatory pathways common to all living animals. The aims of this application are: Specific Aim 1: to optimize electroporation delivery techniques for transgene applications. Specific Aim 2: to create stable transgenic animals using transposon mediated insertion technology. Specific Aim 3: generate transgenic animals expressing loci marked through homologous recombination of BAC clones. Expression constructs have already been generated and solid preliminary data along with personnel with proven history of accomplishment insure that rapid progress will be made. These data will provide the technical basis for gene discovery for important developmental events such as germ layer formation during gastrulation, origin of bilateral symmetry, neural development/organization, and somatic/germ stem cell biology by enhancer trap techniques and for the functional analysis of gene network evolution by insertional mutagenesis. [unreadable] [unreadable] [unreadable]